ノネズミニオケルビョウゲンセイエルシニアニカンスルエキガクテキケンキュウナラビニコレラビョウゲンタイノジンソクケンシュツホウノカイハツ

博士（獣医学）東京農工大

Development and Assessment of a Diagnostic DNA Oligonucleotide Microarray for Detection and Typing of Meningitis-Associated Bacterial Species.

Meningitis is commonly caused by infection with a variety of bacterial or viral pathogens. Acute bacterial meningitis (ABM) can cause severe disease, which can progress rapidly to a critical life-threatening condition. Rapid diagnosis of ABM is critical, as this is most commonly associated with severe sequelae with associated high mortality and morbidity rates compared to viral meningitis, which is less severe and self-limiting. We have designed a microarray for detection and diagnosis of ABM. This has been validated using randomly amplified DNA targets (RADT), comparing buffers with or without formamide, in glass slide format or on the Alere ArrayTubeTM (Alere Technologies GmbH) microarray platform. Pathogen-specific signals were observed using purified bacterial nucleic acids and to a lesser extent using patient cerebral spinal fluid (CSF) samples, with some technical issues observed using RADT and glass slides. Repurposing the array onto the Alere ArrayTubeTM platform and using a targeted amplification system increased specific and reduced nonspecific hybridization signals using both pathogen nucleic and patient CSF DNA targets, better revealing pathogen-specific signals although sensitivity was still reduced in the latter. This diagnostic microarray is useful as a laboratory diagnostic tool for species and strain designation for ABM, rather than for primary diagnosis

Rapid detection and identification of foodborne pathogens using genomics

Infectious diseases caused by Salmonella, Shigella and Shiga toxin-producing E. coli (STEC) place a heavy burden on human health and incur a massive economic cost. Timely detection and identification of these bacterial pathogens is vital for food safety and public health surveillance. Existing detection methods cannot easily distinguish different serotypes of these pathogens and are time-consuming. Early detection and identification of Salmonella, Shigella and STEC can be achieved by detection of highly specific and discriminatory pathogen genomic targets. Thus, comparative genomic analysis of many publicly available genomic sequences of Salmonella, Shigella and STEC has been applied to identify pathogen type-specific gene markers for rapid, highly sensitive and specific identification and differentiation of Salmonella, Shigella and STEC. In this thesis, pathogen type-specific gene markers for Salmonella, Shigella and STEC have been identified through comparative genomic analysis of pathogen genome sequences. For Salmonella, a set of 131 serovar-specific genes were identified for prediction of the 106 common serovars from genomic data with 95.3% accuracy. Seven laboratory diagnostic MCDA assays targeting seven Salmonella serovar-specific genes were then developed for the detection of five most prevalent Salmonella serovars in Australia with high specificity (>93.3%) and high sensitivity (>92.9%). These assays are rapid and can produce results in as short as 8 minutes. For Shigella, cluster-specific genes were identified for differentiation of Shigella and enteroinvasive E. coli (EIEC) from genomic data with 99.64% accuracy and were used to develop an in silico pipeline, ShigEiFinder for accurate differentiation, cluster typing and serotyping of Shigella and EIEC with 99.38% accuracy. For STEC, cluster/serotype-specific genes were identified for typing of STEC with 99.54% accuracy and were used to develop an in silico pipeline, STECFinder which can assign STEC isolates to STEC clusters and serotypes with 99.83% accuracy. These markers could be adapted for metagenomics or culture independent typing and could also be useful in the development of more cost-effective molecular assays. The outcome of this thesis can be applied to rapid typing of respective pathogens in food, clinical and environmental samples and facilitate surveillance of these pathogens for public health control and preventio

A Low-Cost Genomic Sensor for Ocean-Observing Systems and Infectious Disease Detection

abstract: Many environmental microorganisms such as marine microbes are un-culturable; hence, they should be analyzed in situ. Even though a few in situ ocean observing instruments have been available to oceanographers, their applications are limited, because these instruments are expensive and power hungry. In this dissertation project, an inexpensive, portable, low-energy consuming, and highly quantitative microbiological genomic sensor has been developed for in situ ocean-observing systems. A novel real-time colorimetric loop-mediated isothermal amplification (LAMP) technology has been developed for quantitative detection of microbial nucleic acids. This technology was implemented on a chip-level device with an embedded inexpensive imaging device and temperature controller to achieve quantitative detection within one hour. A bubble-free liquid handling approach was introduced to avoid bubble trapping during liquid loading, a common problem in microfluidic devices. An algorithm was developed to reject the effect of bubbles generated during the reaction process, to enable more accurate nucleic acid analysis. This genomic sensor has been validated at gene and gene expression levels using Synechocystis sp. PCC 6803 genomic DNA and total RNA. Results suggest that the detection limits reached 10 copies/μL and 100 fg/μL, respectively. This approach was highly quantitative, with linear standard curves down to 103 copies/μL and 1 pg/μL, respectively. In addition to environmental microbe characterization, this genomic sensor has been employed for viral RNA quantification during an infectious disease outbreak. As the Zika fever was spreading in America, a quantitative detection of Zika virus has been performed. The results show that the genomic sensor is highly quantitative from 10 copies/μL to 105 copies/μL. This suggests that the novel nucleic acid quantification technology is sensitive, quantitative, and robust. It is a promising candidate for rapid microbe detection and quantification in routine laboratories. In the future, this genomic sensor will be implemented in in situ platforms as a core analytical module with minor modifications, and could be easily accessible by oceanographers. Deployment of this microbial genomic sensor in the field will enable new scientific advances in oceanography and provide a possible solution for infectious disease detection.Dissertation/ThesisDoctoral Dissertation Biological Design 201

Molecular-Based Methods to Detect Viable Bacterial Pathogens in Source Waters

Humans can be exposed to waterborne bacterial pathogens and numerous outbreaks have been reported involving these microorganisms around the world. Many different enteric pathogens can be found in source waters used for drinking water. Assessing these pathogens and their possible threat to public health has always been important. Waterborne pathogens can be difficult to detect, and despite a large variety of recognized microbial detection techniques, the cause of many outbreaks has not been unidentified. Effective and rapid pathogen detection techniques are required to achieve reliable data for microbial source water quality, outbreak investigations, and for drinking water treatment efficacy monitoring. Bacteria have long been detected using classical culture-based methods, with the rationale that living cells are able to grow/replicate. However, many pathogenic bacteria in source waters may turn into viable but not culturable (VBNC) cells and are thus undetectable by growth-based methodologies. Alternatively, PCR-based techniques have been developed to detect both non-culturable and culturable bacteria. Yet with these techniques, post-death DNA persistency can inaccurately overestimate the number of viable cells. This problem may be circumvented by an alteration to the PCR procedure that is reported to be able to block PCR amplification of DNA that originates from dead cells. This alteration involves a chemical pre-treatment step prior to PCR using a photoreactive intercalating dye, propidium monoazide (PMA). In this research, a successful modification was made to the PMA-PCR method that can result in substantial suppression of the PCR signal from dead cells, and provide results that can more accurately measure bacterial pathogen viability. PMA-PCR was applied to high concentrations (1 × 107 cells mL-1) of heat-killed cells of Salmonella enterica and Campylobacter jejuni. Using PMA-PCR in combination with primers that amplified a relatively short fragment of the S. enterica invA gene (119 bp), only a 3-log reduction of the dead cell PCR signal was obtained. Similarly, for C. jejuni using PCR primers that amplified a relatively short fragment of DNA (174 bp of cpn60 gene), only a 1-log reduction of the PCR signal was observed for dead cells. Therefore, PMA treatment followed by PCR amplification of short DNA fragments resulted in incomplete signal inhibition of heat killed Salmonella and Campylobacter. To further investigate how PCR conditions can affect the ability of PMA to inhibit PCR amplification, primers were then used that could amplify a larger fragment of DNA. PCR amplification of a longer DNA fragment (1614 bp of invA gene for S. enterica and 1512 bp of cpn60 gene for C. jejuni) strongly suppressed the signal (7 log reduction) for both heat-killed Salmonella and Campylobacter. For UV-treated S. enterica and C. jejuni, short amplicon PMA-PCR showed no or very low PCR signal reduction, in part due to intact membranes directly after UV irradiation. Long amplicon qPCR, however, resulted in dead cell signal removal and PMA pretreatment had no effect on PCR signal suppression. This study used quantitative PCR and the PMA-PCR viability assays to evaluate the levels and occurrences of four groups of pathogenic bacteria in surface water samples from two locations on the Grand River, Ontario, Canada, to demonstrate the reliability of the PMA-PCR technique for the enumeration of viable cells. The bacterial groups investigated included S. enterica, thermophilic Campylobacter, Escherichia coli O157:H7, and Arcobacter butzleri. Small numbers of dead cells (not more than 0.5 log 100 mL-1) were present, detected as the difference between PMA-PCR and PCR without PMA treatment. In this particular river, pathogen enumeration by PCR was only slightly influenced by false positive signal detection due to the presence of dead cells or extracellular DNA and reliable bacterial pathogen detection could be attained by PCR without PMA pretreatment. Viable A. butzleri were detected at elevated concentrations (up to 4.8 log cells per 100 mL) in the Grand River. Arcobacter has not been previously studied in the Grand River and this is one of the few studies that have quantitatively assessed Arcobacter in the environment. This suggests that additional research is required on the pathogenicity of this organism and its occurrence in water. In the next stage of this research, both the improved viability assay (long amplicon PMA-PCR) and conventional quantitative PCR were applied to investigate the survival trends of selected enteric bacterial pathogens including Yersinia enterocolitica, S. enterica, C. jejuni, and A. butzleri. The target bacteria were inoculated into sterile or non-sterile river water to study the impact of background microbiota on cell survival. These experiments were perfomed at 3 different temperatures (5, 15, and 25°C) and at high/low dissolved oxygen (DO) concentrations (for C. jejuni, and A. butzleri only) to evaluate the effect of these potential environmental stresses on bacterial survival trends. The results indicated that the autochthonous microbiota in river water had a significant effect on the bacterial die-off. Although lower temperatures enhanced bacterial survival in non-sterile river water, it was found that PCR may overestimate the effect of temperature on survival and that the PCR viability assays (PMA-PCR) could more accurately measure the impact of temperature. The survival of viable C. jejuni was adversely affected by high DO levels only at a low temperature (5°C) and this effect was observed only when the PMA-PCR viability assay was applied. A. butzleri survival was not affected by water DO levels. This research provides an improved understanding of viable/active enteric waterborne bacteria and their survival in the aquatic microcosms as well as reliable data to better elucidate the effect of environmental factors on the occurrence of pathogenic bacteria. It can also offer valuable information for microbial risk assessments used by regulators and decision makers.1 yea

New Insight into Brucella Infection and Foodborne Diseases

Brucellosis is an important zoonotic disease. More than half a million new cases from 100 countries are reported annually to the World Health Organization (WHO). The majority of patients are living in developing countries. Brucellosis is a systemic infection with a broad clinical spectrum, ranging from an asymptomatic disease to a severe and fatal illness. Clinical and laboratory features vary widely. The main presentations are acute febrile illness, localized infection, and chronic infection. Laboratory tools for diagnosis of brucellosis include culture, serology, and polymerase chain reaction (PCR). The goal of brucellosis therapy is to control the illness and prevent complications, relapses, and sequelae. Important principles of brucellosis treatment include use of antibiotics with activity in the acidic intracellular environment, use of combination regimens, and prolonged duration of treatment. This book is the result of several months of outstanding efforts by the authors and the revision of the content by experts in the field of brucellosis. This book is a valid resource and is intended for everyone interested in infectious disease to learn the most important aspects of brucellosis

Population Dynamics of Enteric Salmonella in Response to Antibiotic Use in Feedlot Cattle

The various uses of antibiotics in feedlot cattle have been a concern as a potential source of antibiotic resistant Salmonella infections in humans. A 26-day randomized controlled longitudinal field trial was undertaken to assess the effects of injectable ceftiofur crystalline-free acid (CCFA) versus in-feed chlortetracycline (CTC) on the temporal dynamics of Salmonella enterica subsp. enterica in feedlot cattle. Two replicates of 8 pens (total of 176 steers) received one of 4 different treatment regimens. All, or one, out of 11 steers were treated with CCFA on day 0 in 8 pens, with half of the pens later receiving three 5-day regimens of CTC. We isolated Salmonella from fecal samples, and antimicrobial susceptibility was assessed. Salmonella in the feces were quantified with probe real-time qPCR targeting invA gene and by direct spiral plating on brilliant green agar. Whole-genome sequencing was performed for all Salmonella isolates to analyze serotype, resistance genotype, MLST, and to explore the phylogenetic relations of the isolates. The mean Salmonella prevalence was 75.0% on day 0, and most isolates were pansusceptible to 14 antibiotics. Both CCFA and CTC reduced the overall prevalence of Salmonella; however, these treatments increased the proportion of multi-drug resistant (MDR) Salmonella. Ceftriaxone and tetracycline resistant Salmonella were detectable in day 0 samples, suggesting that resistant Salmonella existed in the population before antibiotics use. The quantity of resistant Salmonella remained at approximately 10^3 CFU / gram of feces throughout the study. Significantly (P < 0.05) more animals were detected with resistant Salmonella following antibiotic treatments. Among the six serotypes detected, all S. Reading isolates were MDR and carrying an IncA/C2 plasmid, suggesting a strong association between serotype and resistance type. The S. Reading isolates consisted of 2 phylogenetic clades with differential selection by CCFA versus CTC (alone). Our study demonstrated that the selection pressures of a 3rd generation cephalosporin and of CTC during the cattle feeding period selects for antibiotic resistant Salmonella and increases the proportion of cattle carrying resistant Salmonella, even after the treatment period ends. Further investigations are needed to assess whether an extended feeding period of 150 days provides a sufficient ‘wash-out’ period for the gut microbiota to return to normal status

Detection and serotyping of foot-and-mouth disease virus with laboratory and in silico methods

Foot-and-mouth disease virus (FMDV) is a highly contagious animal pathogen and it has a variable genome and high antigenic variation. There are seven known serotypes of this virus: A, O, C, Asia1, SAT1, SAT2, and SAT3. The rapid detection and serotype characterization of the virus is instrumental for the prompt response by animal health authorities. This thesis presents the design and development of the first electronic microarray assay for the simultaneous detection and subtyping of FMDV. The assay was evaluated in silico and it was tested with 19 synthetic DNA constructs representing all 7 serotypes, followed by the testing with 23 viral RNA samples representing all 7 serotypes. Also, various in silico methods were compared for the classification of FMDV sequences using complete genomes and next generation sequencing (NGS) data. Finally, highly specific and highly sensitive single nucleotide variant signatures that distinguish the seven FMDV serotypes were discovered.Chemical, Biological, Radiological-Nuclear, and Explosives Research and Technology Initiative (CRTI) Project 09-403T

Trends in Infectious Diseases

This book gives a comprehensive overview of recent trends in infectious diseases, as well as general concepts of infections, immunopathology, diagnosis, treatment, epidemiology and etiology to current clinical recommendations in management of infectious diseases, highlighting the ongoing issues, recent advances, with future directions in diagnostic approaches and therapeutic strategies. The book focuses on various aspects and properties of infectious diseases whose deep understanding is very important for safeguarding human race from more loss of resources and economies due to pathogens

Microbial Community Analysis of Restaurant Cutting Boards in Seri Kembangan，Malaysia and the Identification of Factors Associated with Foodborne Bacteria Growth

九州工業大学博士学位論文 学位記番号:生工博甲第258号 学位授与年月日:平成28年3月25日1: INTRODUCTION||2: LITERATURE REVIEW||3: MATERIALS AND METHODS||4: ANALYSIS OF MICROBIAL COMMUNITY AND FOODBORNE PATHOGENIC BACTERIA AND THEIR CORRELATION WITH GRADES OF FOOD PREMISES||5: ECOLOGICAL RELATIONSHIP OF MICROBIAL COMMUNITY AND FACTORS ASSOCIATED WITH THE PRESENCE OF FOODBORNE BACTERIA||6: CONCLUDING REMARKS, AND RECOMMENDATIONS FOR FUTURE RESEARCHFoodborne diseases have been associated with microorganisms like bacteria, fungi, viruses and parasites. Most commonly, the outbreaks take place due to the ingestion of pathogenic bacteria like Salmonella typhi, Escherichia coli, Staphylococcus aureus, Vibrio cholerae, Campylobacter jejuni, and Listeria monocytogens. The disease usually happens as a result of toxin secretion of the microorganisms in the intestinal tract of the infected person. Usually, the level of hygiene in the food premises reflect the quality of the food item, hence restaurant or stall with poor sanitary condition is said to be the contributor to food poisoning outbreak. In Malaysia, food poisoning cases are not rare because the hot and humid climate of this country is very suitable for the growth of the foodborne bacteria, thus the government implements strict rules to ensure workers and owners of food premises prioritize the cleanliness of their working area. Studies of food safety are important, and the results can give information about the types of microorganisms, and factors that affect their growth. The result is crucial to determine how the spread of foodborne bacteria can be controlled safely and the outbreak can be reduced. This study adopts the pyrosequencing technique to identify bacteria present on 26 kitchen cutting boards collected from different grades of food premises around Seri Kembangan, Malaysia. The analysis generated 452,401 of total reads of OTUs with an average of 1.4 × 107 bacterial cells/cm2. Proteobacteria, Firmicutes and Bacteroidetes were identified as the most abundant phyla in the samples. Taxonomic richness was generally high with >1000 operational taxonomic units (OTUs) observed across all samples. The highest appearance frequencies (100%) were OTUs closely related to Enterobacter sp., E. aerogenes, Pseudomonas sp. and P. putida. Several OTUs were identified most closely related to known foodborne pathogens including Bacillus cereus, Cronobacter sakazaki, C. turisensis, Escherichia coli, E. coli O157: H7, Salmonella bongori, S. enterica, S. paratyphi, S. typhi, S. typhimurium and Yersinia enterocolitica ranging from 0.005- 0.68% relative abundance. The condition and grade of the food premises on a three-point cleanliness scale did not correlate with the bacterial abundance and type. To add, the community composition of FBB based on premise grades also showed no similarity. Regardless of the status and grades, all food premises have the same likelihood to introduce foodborne bacteria from cutting boards to their foods and must always prioritize the correct food handling procedure, in order to avoid an unwanted outbreak of foodborne illnesses. This study further analyzes the effect that might be created by other microorganisms in order to support or suppress the number of pathogenic bacteria. The analysis found that high bacteria group contains a low percentage of FBB compared to low bacteria group. The results also showed that although premise grade C contains significantly higher numbers of overall bacteria, it did not influence the number of FBB. The combinations of Clostridiales, Flavobacteriales, and Lactobacillales in the high bacteria group were found to have significant negative associations with FBB. Despite the finding that Citrobacter, Enterobacter, Erwinia, Klebsiella and Pantoea had significant positive correlation with FBB, the population of these bacteria was small. This study suggested that high background biota and a combination of different bacteria have an advantage in preventing FBB from reaching the infectious dose level thus, considered harmless